The rabies virus phosphoprotein P [Elektronische Ressource] : a key regulator of innate immune responses / vorgelegt von Krzysztof Brzózka
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The rabies virus phosphoprotein P [Elektronische Ressource] : a key regulator of innate immune responses / vorgelegt von Krzysztof Brzózka

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The rabies virus phosphoprotein P: a key regulator of innate immune responses Dissertation der Fakultät für Biologie der Ludwig-Maximilians-Universität München zur Erlangung des Dr. rer. nat. vorgelegt von Krzysztof Brzózka München, April 2006 Erstgutachter: PD Dr. Bettina Kempkes Zweitgutachter: Prof. Dr. Michael Boshart Sondergutachter: Prof. Dr. Karl-Klaus Conzelmann Tag der mündlichen Prüfung: 22 November 2006 TABLE OF CONTENTS 1 TABLE OF CONTENTS 1 TABLE OF CONTENTS ....................................................................................................................... 3 2 ABBREVIATIONS ................................................................................................................................4 3 INTRODUCTION... 6 3.1 RABIES VIRUS (RV) ........................................................................................................................ 6 3.1.1 Pathogenicity ......................................................................................................................... 6 3.1.2 Virus structure and replication............................................................................................. 7 3.1.3 Multifunctional phosphoprotein P ....................................................................................... 9 3.2 INNATE IMMUNITY: INTERFERON ...........................

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Publié le 01 janvier 2006
Nombre de lectures 48
Poids de l'ouvrage 3 Mo

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  The rabies virus phosphoprotein P: a key
regulator of innate immune respo
    Dissertation der Fakultät für Biologie der Ludwig-Maximilians-Universität München zur Erlangung des Dr. rer. nat.  
 vorgelegt von Krzysztof Brzózka München, April 2006
nses
                         
 Erstgutachter:  Zweitgutachter:  Sondergutachter:  
   
PD Dr. Bettina Kempkes Prof. Dr. Michael Boshart Prof. Dr. Karl-Klaus Conzelmann
Tag der mündlichen Prüfung: 22 November 2006
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TABLE OF CONTENTS
TABLE OF CONTENTS
TABLE OF CONTENTS ....................................................................................................................... 3
ABBREVIATIONS ................................................................................................................................ 4
INTRODUCTION .................................................................................................................................. 6
3.1 RABIES VIRUS(RV) ........................................................................................................................ 6 3.1.1 Pathogenicity ......................................................................................................................... 6 3.1.2 Virus structure and replication............................................................................................. 7 3.1.3 Multifunctional phosphoprotein P ....................................................................................... 9 3.2 INNATE IMMUNITY: INTERFERON.................................................................................................... 11 3.2.1 Interferon induction ............................................................................................................. 11 3.2.1.1 Interferon regulatory factors...................................................................................... 13 3.2.1.2 RNA-helicase pathway................................................................................................ 14 3.2.1.3 Toll-like receptor 3 pathway....................................................................................... 15 3.2.1.4 Toll-like receptor 7/9 pathway.................................................................................... 16 3.2.2 Interferon signaling ............................................................................................................. 17 3.2.3 Interferon stimulated genes (ISGs) .................................................................................... 19 3.3 VIRAL INHIBITORS OF INNATE IMMUNE RESPOSE............................................................................. 20 3.3.1 Viral inhibitors of IFN induction ......................................................................................... 20 3.3.2 Viral inhibitors of interferon signaling .............................................................................. 21
DISCUSSION......................................................................................................................................23 
4.1 4.2 4.3
INHIBITION OF INTERFERON BETA INDUCTION BYRABIES VIRUSPPROTEIN...................................... 23 INHIBITION OF INTERFERON SIGNALING BYRABIES VIRUSPPROTEIN............................................... 27 CONCLUDING REMARKS................................................................................................................ 32
SUMMARY ......................................................................................................................................... 34
ZUSAMMENFASSUNG ..................................................................................................................... 36
REFERENCE LIST.............................................................................................................................38 
APPENDIX: ........................................................................................................................................ 48
8.1 8.2 8.3
ARTICLES ENCLOSED AS A PART OF THE CUMULATIVE DOCTORAL THESIS: ...................................... 48 ACKNOWLEDGEMENTS................................................................................................................. 49 CURRICULUMVITAE..................................................................................................................... 50
2 ABBREVIATIONS AP1 Akt BDV CARD Cardif CBP CNS CpG CRM1 dsRNA dsDNA GAF GAS IFN IFNAR IFNGR IKK IL IPS-1 IRAK ISG ISGF3 ISRE IRF JAK Lgp2 MAVS MDA5 MHC MyD88 NFκB NNSV   2 -5 OAS pDC PAMP PI3K PKR PML RIG-I RNP RSV RV ssRNA SOCS STAT TANK TBK-1 TIR TNF TRAF TRIF
ABBREVIATIONS
activator protein 1 alpha serine/threonine-protein kinase Borna disease virus caspase activation and recruitment domain CARD adapter inducing interferon-beta cAMP-responsive-element-binding protein (CREB)-binding protein central nervous system cytidine-phosphate-guanosine chromosome region maintenance 1 double stranded RNA double stranded DNA gamma-activated factor gamma-activated sequence interferon interferon alpha receptor interferon gamma receptor IκB kinase interleukin interferon-beta promoter stimulator 1 IL-1 receptor associated kinase interferon stimulated gene IFN-stimulated gene factor 3 interferon stimulated response element interferon regulatory factor Janus kinase probable ATP-dependent helicase mitochondrial antiviral signaling protein melanoma differentiation-associated gene 5 major histocompatibility complex myeloid differentiation primary response protein 88 nuclear factor kappa-B non-segmented negative strand RNA viruses 2 -5 oligoadenylate synthetase plasmacytoid dendritic cell pathogen-associated molecular patterns phosphoinositide-3 kinase protein kinase R promyelocytic leukemia protein retinoic acid inducible gene-I ribonucleoprotein respiratory syncytial virus rabies virus single strand RNA suppressor of cytokine signaling signal transducer and activator of transcription TRAF-associated NFκB kinase TANK-binding kinase 1 Toll/IL-1 receptor tumour necrosis factor TNF Receptor-Associated Factor Toll/IL-1R domain-containing adaptor inducing IFN-β 
TRAM TLR VISA VSV VV
TRIF-related adaptor molecule Toll-like receptor virus-induced signaling adaptor vesicular stomatitis virus vaccinia virus
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ABBREVIATIONS
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INTRODUCTION
3.1Rabies virus (RV)  
 INTRODUCTION
3.1.1Pathogenicity   Rabies belongs to one of the oldest known infectious diseases. Already first known reports that are found in Egyptian writings, associate the consequences of a contact with a mad dog with an acute, progressive and incurable encephalitis (Hemachudha et al., 2002; Rupprecht et al., 2002). Rabies is caused by a neurotropic RNA virus of theRhabdoviridaefamily, genusLyssavirus. Bites and scratches represent the typical transmission route of this virus. After initial replication in the peripheral wound RV is transported in a retrograde way to the central nervous system (CNS). The incubation period ranges from one week to several months. The neurotropism of RV is at least in part caused by the use of several receptors in the CNS to facilitate virus entry into neurons: the neural cell adhesion molecule (Thoulouze et al., 1998), the p75 neurotrophin receptor (p75NTR) (Tuffereau et al., 2001; Tuffereau et al., 1998) and the acetylcholine receptor (Lentz et al., 1982). Rabies is characterized by very little neuronal pathology and mild CNS inflammation. Clinical presentation of rabies disease comes in two major forms, encephalitic (furious) and paralytic (dumb), but there is no clear explanation for this dysfunction of the limbic system. Direct post exposure treatment encompasses wound treatment, vaccine administration and inoculation of rabies virus neutralizing immunoglobulins. This treatment is mostly effective when applied in time, but once first symptoms of rabies encephalitis occur, the outcome of the disease is almost always fatal and the management of the disease is palliative (Jackson et al., 2003; Warrell and Warrell, 2004). Recent studies addressed the question of rabies virus pathogenicity and activation of the immune response system. Inflammation and production of neutralizing antibodies, which depend on B lymphocytes and CD4+ T cells, are crucial for clearance of RV from the CNS (Dietzschold, 1993; Dietzschold et al., 1992; Perry and Lodmell,
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 INTRODUCTION
1991; Hooper et al., 1998). As mature neurons are relatively resistant to either cell or cytokine induced cytolysis, the role of cytotoxic CD8+ T cells in RV clearance is
considered minor. A major role can be attributed to cytokine production, mainly IFN-γ. Infection with pathogenic rabies virus results in chemokine production and infiltration of the CNS by mononuclear inflammatory cells (NK cells, T and B lymphocytes). In contrast to attenuated RV, no or only slight activation of innate immune response was observed (Wang et al., 2005; Nakamichi et al., 2004; Nakamichi et al., 2005; Lafon, 2005). Additionally, rabies virus infected neurons retain their integrity but upregulate FasL levels, and thereby induce apoptosis of T-cells, shortly after the T-cells cross the blood-brain barrier (Baloul et al., 2004).  
3.1.2Virus structure and replication   RV is a prototypic virus of thenoMoraviganesel order, the nonsegmented negative strand RNA viruses (NNSV). The RV single strand RNA genome of approximately 12 kb is packed into a bullet-shaped, enveloped virion of approximately 250 nm length and 70 nm width (Figure 1). It comprises five genes encoding nucleoprotein (N), phosphoprotein (P), matrixprotein (M), glycoprotein (G) and polymerase (L) in the order 3-N-P-M-G-L-5. All the five proteins are structural proteins of the virion and are essential for virus replication and spread. The viral RNA is tightly enwrapped by the highly conserved nucleoprotein N and forms a ribonucleoprotein complex (RNP). The RNP serves as a template for the viral polymerase which is composed of a large catalytic subunit L and the polymerase cofactor P. The RNA synthesis, as well as the whole replication cycle, takes place in the cytoplasm. In the transcription mode, a gradient of monocistronic, capped- and polyadenylated mRNAs is synthesized using genomic RNA (-strand) as a template. In this model, the mRNAs of the genes most proximal to the 3 leader promoter are the most abundant ones and the amounts of transcribed mRNAs decrease with the distance from the leader sequence due to dissociation of the polymerase at the gene borders. In the replication mode, full-length RNA is synthesized on both genomic and anti-genomic RNPs (Finke and Conzelmann, 2005). Recombinant rabies viruses can be created from cDNA using a reverse genetics approach. Co-transfection of cDNA expressing the viral
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 INTRODUCTION
anti-genome RNA together with support plasmids expressing rabies virus N, P and L proteins into stable cell line constitutively expressing T7 polymerase (BSR T7/5 cells) results in production of virus particles (Schnell et al., 1994; Buchholz et al., 1999). Recovery of genetically engineered rabies viruses with defined changes in the genome greatly facilitated studies on the different steps of virus life cycle, the determination of viral pathogenicity factors, and on host-virus interactions. The balance between RNP replication and mRNA transcription is regulated by the structural matrix (M) protein (Finke and Conzelmann, 2003; Finke et al., 2003). The M protein, together with the spike glycoprotein (G), is also essential for budding of virus particles. The glycoprotein is the major viral antigen and pathogenicity factor of rabies virus, as shown byin vivoexperiments, involving the exchange of glycoproteins of pathogenic and attenuated rabies virus. Infection with a chimeric virus expressing G protein of an apathogenic strain resulted in abortive infection and increased the survival rate of infected mice (Morimoto et al., 2000; Finke and Conzelmann, 2005). High levels of glycoprotein expression were found to be responsible for induction of apoptosis in infected cells. Downregulation of G expression prevents induction of apoptosis and correlated with virus pathogenicity (Faber et al., 2002; Morimoto et al., 1999; Prehaud et al., 2003; Sarmento et al., 2005).
 
A
B
nucleoprotein N phosphoprotein P matrix protein M glycoprotein G viral polymerase L
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Figure 1.(A) Rabies virus belongs to the family of ssRNA enveloped viruses. The mature rabies virion has a characteristic bullet-shaped appearance of approximately 80 nm diameter and length varying between 130 and 300 nm. (B) Negative stranded RNA genome codes for 5 proteins; nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and polymerase (L). All proteins are structural and can be divided in two groups: a helical ribonucleoprotein core (RNP) and a surrounding envelope. In the RNP, the viral RNA-dependent RNA polymerase consisting of P and L proteins is associated with genomic RNA tightly encased by the N protein. The M protein is associated with both RNP and the envelope and plays an essential role in rabies virus assembly. The rabies glycoprotein forms approximately 400 trimeric spikes which are tightly arranged on the surface of the virus.
 
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